Methods of producing 7-carbon chemicals via c1 carbon chain elongation associated with coenzyme b synthesis

ABSTRACT

This document describes biochemical pathways for producing pimelic acid, 7-aminoheptanoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine or 1,7-heptanediol by forming one or two terminal functional groups, each comprised of carboxyl, amine or hydroxyl group, in a C7 aliphatic backbone substrate. These pathways, metabolic engineering and cultivation strategies described herein rely on the C1 elongation enzymes or homolog associated with coenzyme B biosynthesis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority of U.S. Provisional Application Ser.No. 61/747,406, filed Dec. 31, 2012, and U.S. Provisional ApplicationSer. No. 61/829,088, filed May 30, 2013. The contents of the priorapplications are incorporated herein by reference in their entirety.

TECHNICAL FIELD

This invention relates to methods for biosynthesizing one or more ofpimelic acid, 7-aminoheptanoic acid, heptamethylenediamine and1,7-heptanediol (hereafter “C7 building blocks”) from oxoglutarate oroxiadipate using one or more isolated enzymes such as synthases,dehydratases, hydratases, dehydrogenases, reductases, thioesterases,reversible CoA ligases, CoA transferases, deacetylases, andtransaminases or using recombinant host cells expressing one or moresuch enzymes.

BACKGROUND

Nylons are polyamides which are sometimes synthesized by thecondensation polymerisation of a diamine with a dicarboxylic acid.Similarly, nylons may be produced by the condensation polymerisation oflactams. A ubiquitous nylon is Nylon 6,6, which is produced by reactionof hexamethylenediamine (HMD) and adipic acid. Nylon 6 is produced by aring opening polymerisation of caprolactam (Anton & Baird, PolyamidesFibers, Encyclopedia of Polymer Science and Technology, 2001).

Nylon 7 and Nylon 7,7 represent novel polyamides with value-addedcharacteristics compared to Nylon 6 and Nylon 6,6. Nylon 7 is producedby polymerisation of 7-aminoheptanoic acid, whereas Nylon 7,7 isproduced by condensation polymerisation of pimelic acid andheptamethylenediamine. No economically viable petrochemical routes existto producing the monomers for Nylon 7 and Nylon 7,7.

Given no economically viable petrochemical monomer feedstocks,biotechnology offers an alternative approach via biocatalysis.Biocatalysis is the use of biological catalysts, such as enzymes, toperform biochemical transformations of organic compounds.

Both bioderived feedstocks and petrochemical feedstocks are viablestarting materials for the biocatalysis processes.

Accordingly, against this background, it is clear that there is a needfor methods for producing pimelic acid, 7-aminoheptanoic acid,heptamethylenediamine, 7-hydroxyheptanoic acid and 1,7-heptanediol(hereafter “C7 building blocks”) wherein the methods arebiocatalyst-based.

However, no wild-type prokaryote or eukaryote naturally overproduces orexcretes C7 building blocks to the extracellular environment.Nevertheless, the metabolism of pimelic acid has been reported.

The dicarboxylic acid, pimelic acid, is converted efficiently as acarbon source by a number of bacteria and yeasts via β-oxidation intocentral metabolites. β-oxidation of CoEnzyme A (CoA) activated pimelateto CoA-activated 3-oxopimelate facilitates further catabolism via, forexample, pathways associated with aromatic substrate degradation. Thecatabolism of 3-oxopimeloyl-CoA to acetyl-CoA and glutaryl-CoA byseveral bacteria has been characterized comprehensively (Harwood andParales, Annual Review of Microbiology, 1996, 50, 553-590).

The optimality principle states that microorganisms regulate theirbiochemical networks to support maximum biomass growth. Beyond the needto express heterologous pathways in a host organism, directing carbonflux towards C7 building blocks that serve as carbon sources rather thanto biomass growth constituents, contradicts the optimality principle.For example, transferring the 1-butanol pathway from Clostridium speciesinto other production strains has often fallen short by an order ofmagnitude compared to the production performance of native producers(Shen et al., Appl. Environ. Microbiol., 2011, 77(9), 2905-2915).

The efficient synthesis of the seven carbon aliphatic backbone precursoris a key consideration in synthesizing C7 building blocks prior toforming terminal functional groups, such as carboxyl, amine or hydroxylgroups, on the C7 aliphatic backbone.

SUMMARY

This document is based at least in part on the discovery that it ispossible to construct biochemical pathways for producing a seven carbonchain aliphatic backbone precursor, in which one or two functionalgroups, i.e., carboxyl, amine, or hydroxyl, can be formed, leading tothe synthesis of one or more of pimelic acid, 7-aminoheptanoate,7-hydroxyheptanoate, heptamethylenediamine, and 1,7-heptanediol(hereafter “C7 building blocks). Pimelic acid and pimelate,7-hydroxyheptanoic acid and 7-hydroxyheptanoate, and 7-aminoheptanoicand 7-aminoheptanoate are used interchangeably herein to refer to thecompound in any of its neutral or ionized forms, including any saltforms thereof. It is understood by those skilled in the art that thespecific form will depend on pH. These pathways, metabolic engineeringand cultivation strategies described herein rely on the C1 elongationenzymes or homologs associated with the Coenzyme B biosynthesis pathwayof methanogens.

In the face of the optimality principle, it surprisingly has beendiscovered that appropriate non-natural pathways, feedstocks, hostmicroorganisms, attenuation strategies to the host's biochemical networkand cultivation strategies may be combined to efficiently produce one ormore C7 building blocks.

In some embodiments, the C7 aliphatic backbone for conversion to one ormore C7 building blocks is 7-oxoheptanoate (also known as pimelatesemialdehyde) or pimeloyl-CoA (also known as 6-carboxyhexanoyl-CoA),which can be formed from 2-oxoadipate via two cycles of C1 carbon chainelongation. Pimelate semialdehyde and pimeloyl-CoA also can be formedfrom 2-oxoglutarate via three cycles of C1 carbon chain elongation(2-oxoacid elongation cycles) (i.e., from 2-oxoglutarate to2-oxosuberate, followed by decarboxylation to either pimelatesemialdehyde or pimeloyl-CoA). See FIG. 1.

In some embodiments, a terminal carboxyl group can be enzymaticallyformed using a decarboxylase, a thioesterase, an aldehyde dehydrogenase,a 6-oxohexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, areversible CoA-ligase (e.g., a reversible succinyl-CoA-ligase), or aCoA-transferase (e.g., a glutaconate CoA-transferase). See FIG. 1 andFIG. 2.

In some embodiments, a terminal amine group can be enzymatically formedusing a ω-transaminase or a deacetylase. See FIG. 3 and FIG. 4.

In some embodiments, a terminal hydroxyl group can be enzymaticallyformed using a 4-hydroxybutyrate dehydrogenase, 5-hydroxypentanoatedehydrogenase or a 6-hydroxyhexanoate dehydrogenase or an alcoholdehydrogenase. See FIG. 5 and FIG. 6.

In one aspect, this document features a method for biosynthesizing aproduct selected from the group consisting of pimelic acid,7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine and1,7-heptanediol. The method includes enzymatically synthesizing a sevencarbon chain aliphatic backbone from 2-oxoglutarate via three cycles of2-oxoacid carbon chain elongation and enzymatically forming one or twoterminal functional groups selected from the group consisting ofcarboxyl, amine, and hydroxyl groups in the backbone, thereby formingthe product. The seven carbon chain aliphatic backbone can bepimeloyl-CoA or pimelate semialdehyde. Each of the three cycles of2-oxoacid chain elongation can include using a (homo)_(n)citratesynthase, a (homo)_(n)citrate dehydratase, a (homo)_(n)aconitatehydratase and an iso(homo)_(n)citrate dehydrogenase to form2-oxosuberate from 2-oxoglutarate. The 2-oxosuberate can be converted topimelate semialdehyde by an indolepyruvate decarboxylase or converted topimeloyl-CoA by a 2-oxoglutarate dehydrogenase complex.

The two terminal functional groups can be the same (e.g., amine orhydroxyl) or can be different (e.g., a terminal amine and a terminalcarboxyl group; or a terminal hydroxyl group and a terminal carboxylgroup).

A ω-transaminase or a deacetylase can enzymatically form an amine group.The ω-transaminase can have at least 70% sequence identity to any one ofthe amino acid sequences set forth in SEQ ID NO. 8-13.

A 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase,a 4-hydroxybutyrate dehydratase, or an alcohol dehydrogenase canenzymatically form a hydroxyl group.

A thioesterase, an aldehyde dehydrogenase, a 7-oxoheptanoatedehydrogenase, a 6-oxohexanoate dehydrogenase, a CoA-transferase (e.g. aglutaconate CoA transferase), or a reversible CoA-ligase (e.g., areversible succinate-CoA ligase) can enzymatically forms a terminalcarboxyl group. The thioesterase can have at least 70% sequence identityto the amino acid sequence set forth in SEQ ID NO: 1.

A carboxylate reductase and a phosphopantetheinyl transferase can form aterminal aldehyde group as an intermediate in forming the product. Thecarboxylate reductase can have at least 70% sequence identity to any oneof the amino acid sequences set forth in SEQ ID NO. 2-7.

Any of the methods can be performed in a recombinant host byfermentation. The host can be subjected to a cultivation strategy underanaerobic, micro-aerobic or mixed oxygen/denitrification cultivationconditions. The host can be cultured under conditions of nutrientlimitation. The host can be retained using a ceramic hollow fibermembrane to maintain a high cell density during fermentation. The finalelectron acceptor can be an alternative to oxygen such as nitrates.

In any of the methods, the host's tolerance to high concentrations of aC7 building block can be improved through continuous cultivation in aselective environment.

The principal carbon source fed to the fermentation can derive frombiological or non-biological feedstocks. In some embodiments, thebiological feedstock is, includes, or derives from, monosaccharides,disaccharides, lignocellulose, hemicellulose, cellulose, lignin,levulinic acid and formic acid, triglycerides, glycerol, fatty acids,agricultural waste, condensed distillers' solubles, or municipal waste.

In some embodiments, the non-biological feedstock is or derives fromnatural gas, syngas, CO₂/H₂, methanol, ethanol, benzoate, non-volatileresidue (NVR) or a caustic wash waste stream from cyclohexane oxidationprocesses, or a terephthalic acid/isophthalic acid mixture waste stream.

This document also features a recombinant host that includes at leastone exogenous nucleic acid encoding (i) a (homo)_(n)citrate synthase,(ii) a (homo)_(n)citrate dehydratase and a (homo)_(n)aconitatehydratase, (iii) an iso(homo)_(n)citrate dehydrogenase, and (iv) anindolepyruvate decarboxylase or a 2-oxoglutarate dehydrogenase complex,the host producing pimeloyl-CoA or pimelate semialdehyde.

A recombinant host producing pimeloyl-CoA further can include at leastone exogenous nucleic acid encoding one or more of a thioesterase, analdehyde dehydrogenase, a 7-oxoheptanoate dehydrogenase, a6-oxohexanoate dehydrogenase, a CoA-transferase, a reversible CoA-ligase(e.g., a reversible succinyl-CoA-ligase), an acetylating aldehydedehydrogenase, or a carboxylate reductase, the host producing pimelicacid or pimelate semialdehyde. In any of the recombinant hostsexpressing a carboxylate reductase, a phosphopantetheinyl transferasealso can be expressed to enhance the activity of the carboxylatereductase.

A recombinant host producing pimelate semialdehyde further can includeat least one exogenous nucleic acid encoding a ω-transaminase, andproducing 7-aminoheptanoate.

A recombinant host producing pimelate semialdehyde further can includeat least one exogenous nucleic acid encoding a 4-hydroxybutyratedehydrogenase, a 5-hydroxypentanoate dehydrogenase or a6-hydroxyhexanoate dehydrogenase, the host producing 7-hydroxyheptanoicacid.

A recombinant host producing pimelate semialdehyde, 7-aminoheptanoate,or 7-hydroxyheptanoic acid further can include a carboxylate reductase,a ω-transaminase, a deacetylase, an N-acetyl transferase, or an alcoholdehydrogenase, the host producing heptamethylenediamine.

A recombinant host producing 7-hydroxyheptanoic acid further can includeat least one exogenous nucleic acid encoding a carboxylate reductase oran alcohol dehydrogenase, the host producing 1,7-heptanediol.

The recombinant host can be a prokaryote, e.g., from the genusEscherichia such as Escherichia coli; from the genus Clostridia such asClostridium ljungdahlii, Clostridium autoethanogenum or Clostridiumkluyveri; from the genus Corynebacteria such as Corynebacteriumglutamicum; from the genus Cupriavidus such as Cupriavidus necator orCupriavidus metallidurans; from the genus Pseudomonas such asPseudomonas fluorescens, Pseudomonas putida or Pseudomonas oleavorans;from the genus Delftia acidovorans, from the genus Bacillus such asBacillus subtillis; from the genes Lactobacillus such as Lactobacillusdelbrueckii; from the genus Lactococcus such as Lactococcus lactis orfrom the genus Rhodococcus such as Rhodococcus equi.

The recombinant host can be a eukaryote, e.g., a eukaryote from thegenus Aspergillus such as Aspergillus niger; from the genusSaccharomyces such as Saccharomyces cerevisiae; from the genus Pichiasuch as Pichia pastoris; from the genus Yarrowia such as Yarrowialipolytica, from the genus Issatchenkia such as Issathenkia orientalis,from the genus Debaryomyces such as Debaryomyces hansenii, from thegenus Arxula such as Arxula adenoinivorans, or from the genusKluyveromyces such as Kluyveromyces lactis.

Any of the recombinant hosts described herein further can include one ormore of the following attenuated enzymes: a polymer synthase, aNADPH-specific L-glutamate dehydrogenase, a NADPH-consumingtranshydrogenase, a pimeloyl-CoA dehydrogenase; an acyl-CoAdehydrogenase that degrades C7 building blocks and their precursors; aglutaryl-CoA dehydrogenase; or a pimeloyl-CoA synthetase.

Any of the recombinant hosts described herein further can overexpressone or more genes encoding: a 6-phosphogluconate dehydrogenase; atransketolase; a feedback resistant shikimate kinase; a feedbackresistant 2-dehydro-3-deoxyphosphoheptanate aldose; a puridinenucleotide transhydrogenase; a glyceraldehyde-3P-dehydrogenase; a malicenzyme; a glucose-6-phosphate dehydrogenase; a fructose 1,6diphosphatase; a ferredoxin-NADP reductase, a L-alanine dehydrogenase; aNADH-specific L-glutamate dehydrogenase; a diamine transporter; adicarboxylate transporter; and/or a multidrug transporter.

The reactions of the pathways described herein can be performed in oneor more cell (e.g., host cell) strains (a) naturally expressing one ormore relevant enzymes, (b) genetically engineered to express one or morerelevant enzymes, or (c) naturally expressing one or more relevantenzymes and genetically engineered to express one or more relevantenzymes. Alternatively, relevant enzymes can be extracted from any ofthe above types of host cells and used in a purified or semi-purifiedform. Extracted enzymes can optionally be immobilized to a solidsubstrate such as the floors and/or walls of appropriate reactionvessels. Moreover, such extracts include lysates (e.g., cell lysates)that can be used as sources of relevant enzymes. In the methods providedby the document, all the steps can be performed in cells (e.g., hostcells), all the steps can be performed using extracted enzymes, or someof the steps can be performed in cells and others can be performed usingextracted enzymes.

Many of the enzymes described herein catalyze reversible reactions, andthe reaction of interest may be the reverse of the described reaction.The schematic pathways shown in FIGS. 1-6 illustrate the reaction ofinterest for each of the intermediates.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and the drawings, and from the claims. The word “comprising”in the claims may be replaced by “consisting essentially of” or with“consisting of,” according to standard practice in patent law.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic of an exemplary biochemical pathway leading topimelate semialdehyde or pimeloyl-CoA using the 2-oxoacid chainelongation pathway associated with coenzyme B synthesis and2-oxoglutarate or 2-oxoadipate as a central metabolite.

FIG. 2 is a schematic of exemplary biochemical pathways leading topimelate using pimeloyl-CoA or pimelate semialdehyde as a centralprecursor.

FIG. 3 is a schematic of exemplary biochemical pathways leading to7-aminoheptanoate using pimeloyl-CoA, pimelate or pimelate semialdehydeas a central precursor.

FIG. 4 is a schematic of an exemplary biochemical pathways leading toheptamethylenediamine using 7-aminoheptanoate, 7-hydroxyheptanoate orpimelate semialdehyde as a central precursor.

FIG. 5 is a schematic of exemplary biochemical pathways leading to7-hydroxyheptanoate using pimelate, pimeloyl-CoA or pimelatesemialdehyde as a central precursor.

FIG. 6 is a schematic of an exemplary biochemical pathway leading to1,7-heptanediol using 7-hydroxyheptanoate as a central precursor.

FIG. 7 contains the amino acid sequences of an Escherichia colithioesterase encoded by tesB (See GenBank Accession No. AAA24665.1, SEQID NO: 1), a Mycobacterium marinum carboxylate reductase (See GenbankAccession No. ACC40567.1, SEQ ID NO: 2), a Mycobacterium smegmatiscarboxylate reductase (See Genbank Accession No. ABK71854.1, SEQ ID NO:3), a Segniliparus rugosus carboxylate reductase (See Genbank AccessionNo. EFV11917.1, SEQ ID NO: 4), a Mycobacterium smegmatis carboxylatereductase (See Genbank Accession No. ABK75684.1, SEQ ID NO: 5), aMycobacterium massiliense carboxylate reductase (See Genbank AccessionNo. EIV11143.1, SEQ ID NO: 6), a Segniliparus rotundus carboxylatereductase (See Genbank Accession No. ADG98140.1, SEQ ID NO: 7), aChromobacterium violaceum ω-transaminase (See Genbank Accession No.AAQ59697.1, SEQ ID NO: 8), a Pseudomonas aeruginosa ω-transaminase (SeeGenbank Accession No. AAG08191.1, SEQ ID NO: 9), a Pseudomonas syringaeω-transaminase (See Genbank Accession No. AAY39893.1, SEQ ID NO: 10), aRhodobacter sphaeroides ω-transaminase (see Genbank Accession No.ABA81135.1, SEQ ID NO: 11), an Escherichia coli ω-transaminase (seeGenbank Accession No. AAA57874.1, SEQ ID NO: 12), a Vibrio fluvialisω-transaminase (see Genbank Accession No. AEA39183.1, SEQ ID NO: 13), aBacillus subtilis phosphopantetheinyl transferase (see Genbank AccessionNo. CAA44858.1, SEQ ID NO:14), or a Nocardia sp. NRRL 5646phosphopantetheinyl transferase (see Genbank Accession No. ABI83656.1,SEQ ID NO:15).

FIG. 8 is a bar graph of the relative absorbance at 412 nm after 20minutes of released CoA as a measure of the activity of a thioesterasefor converting pimeloyl-CoA to pimelate relative to the empty vectorcontrol.

FIG. 9 is a bar graph summarizing the change in absorbance at 340 nmafter 20 minutes, which is a measure of the consumption of NADPH andactivity of carboxylate reductases relative to the enzyme only controls(no substrate).

FIG. 10 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and the activityof carboxylate reductases for converting pimelate to pimelatesemialdehyde relative to the empty vector control.

FIG. 11 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and the activityof carboxylate reductases for converting 7-hydroxyheptanoate to7-hydroxyheptanal relative to the empty vector control.

FIG. 12 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and the activityof carboxylate reductases for converting N7-acetyl-7-aminoheptanoate toN7-acetyl-7-aminoheptanal relative to the empty vector control.

FIG. 13 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and activity ofcarboxylate reductases for converting pimelate semialdehyde toheptanedial relative to the empty vector control.

FIG. 14 is a bar graph summarizing the percent conversion after 4 hoursof pyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity of the enzyme only controls (no substrate).

FIG. 15 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity for converting 7-aminoheptanoate to pimelate semialdehyderelative to the empty vector control.

FIG. 16 is a bar graph of the percent conversion after 4 hours ofL-alanine to pyruvate (mol/mol) as a measure of the ω-transaminaseactivity for converting pimelate semialdehyde to 7-aminoheptanoaterelative to the empty vector control.

FIG. 17 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity for converting heptamethylenediamine to 7-aminoheptanalrelative to the empty vector control.

FIG. 18 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity for converting N7-acetyl-1,7-diaminoheptane toN7-acetyl-7-aminoheptanal relative to the empty vector control.

FIG. 19 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity for converting 7-aminoheptanol to 7-oxoheptanol relative to theempty vector control.

DETAILED DESCRIPTION

This document provides enzymes, non-natural pathways, cultivationstrategies, feedstocks, host microorganisms and attenuations to thehost's biochemical network, which generates a seven carbon chainaliphatic backbone from central metabolites in which one or two terminalfunctional groups may be formed leading to the synthesis of pimelicacid, 7-aminoheptanoic acid, heptamethylenediamine or 1,7-heptanediol(referred to as “C7 building blocks” herein). As used herein, the term“central precursor” is used to denote any metabolite in any metabolicpathway shown herein leading to the synthesis of a C7 building block.The term “central metabolite” is used herein to denote a metabolite thatis produced in all microorganisms to support growth.

Host microorganisms described herein can include endogenous pathwaysthat can be manipulated such that one or more C7 building blocks can beproduced. In an endogenous pathway, the host microorganism naturallyexpresses all of the enzymes catalyzing the reactions within thepathway. A host microorganism containing an engineered pathway does notnaturally express all of the enzymes catalyzing the reactions within thepathway but has been engineered such that all of the enzymes within thepathway are expressed in the host.

The term “exogenous” as used herein with reference to a nucleic acid (ora protein) and a host refers to a nucleic acid that does not occur in(and cannot be obtained from) a cell of that particular type as it isfound in nature or a protein encoded by such a nucleic acid. Thus, anon-naturally-occurring nucleic acid is considered to be exogenous to ahost once in the host. It is important to note thatnon-naturally-occurring nucleic acids can contain nucleic acidsubsequences or fragments of nucleic acid sequences that are found innature provided the nucleic acid as a whole does not exist in nature.For example, a nucleic acid molecule containing a genomic DNA sequencewithin an expression vector is non-naturally-occurring nucleic acid, andthus is exogenous to a host cell once introduced into the host, sincethat nucleic acid molecule as a whole (genomic DNA plus vector DNA) doesnot exist in nature. Thus, any vector, autonomously replicating plasmid,or virus (e.g., retrovirus, adenovirus, or herpes virus) that as a wholedoes not exist in nature is considered to be non-naturally-occurringnucleic acid. It follows that genomic DNA fragments produced by PCR orrestriction endonuclease treatment as well as cDNAs are considered to benon-naturally-occurring nucleic acid since they exist as separatemolecules not found in nature. It also follows that any nucleic acidcontaining a promoter sequence and polypeptide-encoding sequence (e.g.,cDNA or genomic DNA) in an arrangement not found in nature isnon-naturally-occurring nucleic acid. A nucleic acid that isnaturally-occurring can be exogenous to a particular host microorganism.For example, an entire chromosome isolated from a cell of yeast x is anexogenous nucleic acid with respect to a cell of yeast y once thatchromosome is introduced into a cell of yeast y.

In contrast, the term “endogenous” as used herein with reference to anucleic acid (e.g., a gene) (or a protein) and a host refers to anucleic acid (or protein) that does occur in (and can be obtained from)that particular host as it is found in nature. Moreover, a cell“endogenously expressing” a nucleic acid (or protein) expresses thatnucleic acid (or protein) as does a host of the same particular type asit is found in nature. Moreover, a host “endogenously producing” or that“endogenously produces” a nucleic acid, protein, or other compoundproduces that nucleic acid, protein, or compound as does a host of thesame particular type as it is found in nature.

For example, depending on the host and the compounds produced by thehost, one or more of the following enzymes may be expressed in the hostin addition to a (homo)_(n)citrate synthase, a (homo)_(n)citratedehydratase, a (homo)_(n)aconitate hydratase and an iso(homo)_(n)citratedehydrogenase: a indolepyruvate decarboxylase or 2-oxoglutaratedehydrogenase complex, a thioesterase, a reversible CoA-ligase (e.g., areversible succinyl-CoA-ligase), a CoA-transferase (e.g., a glutaconateCoA-transferase), an acetylating aldehyde dehydrogenase, a6-oxohexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, analdehyde dehydrogenase, a carboxylate reductase, a ω-transaminase, aN-acetyl transferase, an alcohol dehydrogenase, a deacetylase, a6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase,or a 4-hydroxybutyrate dehydrogenase. In recombinant hosts expressing acarboxylate reductase, a phosphopantetheinyl transferase also can beexpressed as it enhances activity of the carboxylate reductase.

For example, a recombinant host can include at least one exogenousnucleic acid encoding a (homo)_(n)citrate synthase, a (homo)_(n)citratedehydratase, a (homo)_(n)aconitate hydratase, and aniso(homo)_(n)citrate dehydrogenase, a indolepyruvate decarboxylase and a2-oxoglutarate dehydrogenase complex, and produce pimelate semialdehydeor pimeloyl-CoA.

Such recombinant hosts further can include at least one exogenousnucleic acid encoding one or more of a thioesterase, an aldehydedehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoatedehydrogenase, a CoA-transferase, a reversible CoA-ligase, anacetylating aldehyde dehydrogenase, or a carboxylate reductase andproduce pimelic acid or pimelate semialdehyde. For example, arecombinant host producing pimeloyl-[acp] or pimeloyl-CoA further caninclude a thioesterase, a reversible Co-ligase (e.g., a reversiblesuccinyl-CoA ligase), or a CoA transferase (e.g., a glutaconateCoA-transferase) and produce pimelic acid. For example, a recombinanthost producing pimeloyl-CoA further can include an acetylating aldehydedehydrogenase and produce pimelate semilaldehyde. For example, arecombinant host producing pimelate further can include a carboxylatereductase and produce pimelate semialdehyde.

A recombinant hosts producing pimelic acid or pimelate semialdehydefurther can include at least one exogenous nucleic acid encoding aω-transaminase and produce 7-aminoheptanoate. In some embodiments, arecombinant host producing pimeloyl-CoA includes a carboxylate reductaseand a ω-transaminase to produce 7-aminoheptanoate.

A recombinant host producing pimelate or pimelate semialdehyde furthercan include at least one exogenous nucleic acid encoding a6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase ora 4-hydroxybutyrate dehydrogenase, and produce 7-hydroxyheptanoic acid.In some embodiments, a recombinant host producing pimeloyl-CoA includesan acetylating aldehyde dehydrogenase, and a 6-hydroxyhexanoatedehydrogenase, a 5-hydroxypentanoate dehydrogenase or a4-hydroxybutyrate dehydrogenase to produce 7-hydroxyheptanoate. In someembodiments, a recombinant host producing pimelate includes acarboxylate reductase and a 6-hydroxyhexanoate dehydrogenase, a5-hydroxypentanoate dehydrogenase or a 4-hydroxybutyrate dehydrogenaseto produce 7-hydroxyheptanoate.

A recombinant hosts producing 7-aminoheptanoate, 7-hydroxyheptanoate orpimelate semialdehyde further can include at least one exogenous nucleicacid encoding a ω-transaminase, a deacetylase, a N-acetyl transferase,or an alcohol dehydrogenase, and produce heptamethylenediamine. Forexample, a recombinant host producing 7-hydroxyheptanoate can include acarboxylate reductase with a phosphopantetheine transferase enhancer, aω-transaminase and an alcohol dehydrogenase.

A recombinant host producing 7-hydroxyheptanoic acid further can includeone or more of a carboxylate reductase with a phosphopantetheinetransferase enhancer and an alcohol dehydrogenase, and produce1,7-heptanediol.

Within an engineered pathway, the enzymes can be from a single source,i.e., from one species or genus, or can be from multiple sources, i.e.,different species or genera. Nucleic acids encoding the enzymesdescribed herein have been identified from various organisms and arereadily available in publicly available databases such as GenBank orEMBL.

Any of the enzymes described herein that can be used for production ofone or more C7 building blocks can have at least 70% sequence identity(homology) (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or100%) to the amino acid sequence of the corresponding wild-type enzyme.It will be appreciated that the sequence identity can be determined onthe basis of the mature enzyme (e.g., with any signal sequence removed).

For example, a thioesterase described herein can have at least 70%sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100%) to the amino acid sequence of an Escherichiacoli thioesterase encoded by tesB (see GenBank Accession No. AAA24665.1,SEQ ID NO: 1). See FIG. 7.

For example, a carboxylate reductase described herein can have at least70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%,95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of aMycobacterium marinum (see Genbank Accession No. ACC40567.1, SEQ ID NO:2), a Mycobacterium smegmatis (see Genbank Accession No. ABK71854.1, SEQID NO: 3), a Segniliparus rugosus (see Genbank Accession No. EFV11917.1,SEQ ID NO: 4), a Mycobacterium smegmatis (see Genbank Accession No.ABK75684.1, SEQ ID NO: 5), a Mycobacterium massiliense (see GenbankAccession No. EIV11143.1, SEQ ID NO: 6), or a Segniliparus rotundus (seeGenbank Accession No. ADG98140.1, SEQ ID NO: 7) carboxylate reductase.See, FIG. 7.

For example, a ω-transaminase described herein can have at least 70%sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100%) to the amino acid sequence of a Chromobacteriumviolaceum (see Genbank Accession No. AAQ59697.1, SEQ ID NO: 8), aPseudomonas aeruginosa (see Genbank Accession No. AAG08191.1, SEQ ID NO:9), a Pseudomonas syringae (see Genbank Accession No. AAY39893.1, SEQ IDNO: 10), a Rhodobacter sphaeroides (see Genbank Accession No.ABA81135.1, SEQ ID NO: 11), an Escherichia coli (see Genbank AccessionNo. AAA57874.1, SEQ ID NO: 12), or a Vibrio fluvialis (see GenbankAccession No. AEA39183.1, SEQ ID NO: 13) ω-transaminase. Some of thesew-transaminases are diamine ω-transaminases.

For example, a phosphopantetheinyl transferase described herein can haveat least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%,90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of aBacillus subtilis phosphopantetheinyl transferase (see Genbank AccessionNo. CAA44858.1, SEQ ID NO:14) or a Nocardia sp. NRRL 5646phosphopantetheinyl transferase (see Genbank Accession No. ABI83656.1,SEQ ID NO:15). See FIG. 7.

The percent identity (homology) between two amino acid sequences can bedetermined as follows. First, the amino acid sequences are aligned usingthe BLAST 2 Sequences (Bl2seq) program from the stand-alone version ofBLASTZ containing BLASTP version 2.0.14. This stand-alone version ofBLASTZ can be obtained from Fish & Richardson's web site (e.g.,www.fr.com/blast/) or the U.S. government's National Center forBiotechnology Information web site (www.ncbi.nlm.nih.gov). Instructionsexplaining how to use the Bl2seq program can be found in the readme fileaccompanying BLASTZ. Bl2seq performs a comparison between two amino acidsequences using the BLASTP algorithm. To compare two amino acidsequences, the options of Bl2seq are set as follows: -i is set to a filecontaining the first amino acid sequence to be compared (e.g.,C:\seq1.txt); -j is set to a file containing the second amino acidsequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o isset to any desired file name (e.g., C:\output.txt); and all otheroptions are left at their default setting. For example, the followingcommand can be used to generate an output file containing a comparisonbetween two amino acid sequences: C:\Bl2seq -i c:\seq1.txt -jc:\seq2.txt -p blastp -o c:\output.txt. If the two compared sequencesshare homology (identity), then the designated output file will presentthose regions of homology as aligned sequences. If the two comparedsequences do not share homology (identity), then the designated outputfile will not present aligned sequences. Similar procedures can befollowing for nucleic acid sequences except that blastn is used.

Once aligned, the number of matches is determined by counting the numberof positions where an identical amino acid residue is presented in bothsequences. The percent identity (homology) is determined by dividing thenumber of matches by the length of the full-length polypeptide aminoacid sequence followed by multiplying the resulting value by 100. It isnoted that the percent identity (homology) value is rounded to thenearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is roundeddown to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded upto 78.2. It also is noted that the length value will always be aninteger.

It will be appreciated that a number of nucleic acids can encode apolypeptide having a particular amino acid sequence. The degeneracy ofthe genetic code is well known to the art; i.e., for many amino acids,there is more than one nucleotide triplet that serves as the codon forthe amino acid. For example, codons in the coding sequence for a givenenzyme can be modified such that optimal expression in a particularspecies (e.g., bacteria or fungus) is obtained, using appropriate codonbias tables for that species.

Functional fragments of any of the enzymes described herein can also beused in the methods of the document. The term “functional fragment” asused herein refers to a peptide fragment of a protein that has at least25% (e.g., at least: 30%; 40%; 50%; 60%; 70%; 75%; 80%; 85%; 90%; 95%;98%; 99%; 100%; or even greater than 100%) of the activity of thecorresponding mature, full-length, wild-type protein. The functionalfragment can generally, but not always, be comprised of a continuousregion of the protein, wherein the region has functional activity.

This document also provides (i) functional variants of the enzymes usedin the methods of the document and (ii) functional variants of thefunctional fragments described above. Functional variants of the enzymesand functional fragments can contain additions, deletions, orsubstitutions relative to the corresponding wild-type sequences. Enzymeswith substitutions will generally have not more than 50 (e.g., not morethan one, two, three, four, five, six, seven, eight, nine, ten, 12, 15,20, 25, 30, 35, 40, or 50) amino acid substitutions (e.g., conservativesubstitutions). This applies to any of the enzymes described herein andfunctional fragments. A conservative substitution is a substitution ofone amino acid for another with similar characteristics. Conservativesubstitutions include substitutions within the following groups: valine,alanine and glycine; leucine, valine, and isoleucine; aspartic acid andglutamic acid; asparagine and glutamine; serine, cysteine, andthreonine; lysine and arginine; and phenylalanine and tyrosine. Thenonpolar hydrophobic amino acids include alanine, leucine, isoleucine,valine, proline, phenylalanine, tryptophan and methionine. The polarneutral amino acids include glycine, serine, threonine, cysteine,tyrosine, asparagine and glutamine. The positively charged (basic) aminoacids include arginine, lysine and histidine. The negatively charged(acidic) amino acids include aspartic acid and glutamic acid. Anysubstitution of one member of the above-mentioned polar, basic or acidicgroups by another member of the same group can be deemed a conservativesubstitution. By contrast, a nonconservative substitution is asubstitution of one amino acid for another with dissimilarcharacteristics.

Deletion variants can lack one, two, three, four, five, six, seven,eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidsegments (of two or more amino acids) or non-contiguous single aminoacids. Additions (addition variants) include fusion proteins containing:(a) any of the enzymes described herein or a fragment thereof; and (b)internal or terminal (C or N) irrelevant or heterologous amino acidsequences. In the context of such fusion proteins, the term“heterologous amino acid sequences” refers to an amino acid sequenceother than (a). A heterologous sequence can be, for example a sequenceused for purification of the recombinant protein (e.g., FLAG,polyhistidine (e.g., hexahistidine), hemagglutinin (HA),glutathione-S-transferase (GST), or maltosebinding protein (MBP)).Heterologous sequences also can be proteins useful as detectablemarkers, for example, luciferase, green fluorescent protein (GFP), orchloramphenicol acetyl transferase (CAT). In some embodiments, thefusion protein contains a signal sequence from another protein. Incertain host cells (e.g., yeast host cells), expression and/or secretionof the target protein can be increased through use of a heterologoussignal sequence. In some embodiments, the fusion protein can contain acarrier (e.g., KLH) useful, e.g., in eliciting an immune response forantibody generation) or ER or Golgi apparatus retention signals.Heterologous sequences can be of varying length and in some cases can bea longer sequences than the full-length target proteins to which theheterologous sequences are attached.

Engineered hosts can naturally express none or some (e.g., one or more,two or more, three or more, four or more, five or more, or six or more)of the enzymes of the pathways described herein. Thus, a pathway withinan engineered host can include all exogenous enzymes, or can includeboth endogenous and exogenous enzymes. Endogenous genes of theengineered hosts also can be disrupted to prevent the formation ofundesirable metabolites or prevent the loss of intermediates in thepathway through other enzymes acting on such intermediates. Engineeredhosts can be referred to as recombinant hosts or recombinant host cells.As described herein recombinant hosts can include nucleic acids encodingone or more of a synthase, a dehydratase, a hydratase, a dehydrogenase,a thioesterase, a reversible CoA-ligase, a CoA-transferase, a reductase,deacetylase, N-acetyl transferase or a ω-transaminase as described inmore detail below.

In addition, the production of one or more C7 building blocks can beperformed in vitro using the isolated enzymes described herein, using alysate (e.g., a cell lysate) from a host microorganism as a source ofthe enzymes, or using a plurality of lysates from different hostmicroorganisms as the source of the enzymes.

Enzymes Generating the C7 Aliphatic Backbone for Conversion to C7Building Blocks

As depicted in FIG. 1, a C7 aliphatic backbone for conversion to a C7building block can be formed from 2-oxoglutarate via three cycles of2-oxoacid carbon chain elongation associated with Coenzyme Bbiosynthesis enzymes. A C7 aliphatic backbone for conversion to a C7building block also can be formed from 2-oxoadipate via two cycles of C1carbon chain elongation associated with Coenzyme B biosynthesis enzymes.

In some embodiments, a C1 carbon chain elongation cycle comprises asynthetase, a dehydratase, a dehydratase, and a dehydrogenase. Forexample, each elongation cycle can use a (homo)_(n)citrate dehydratase,a (homo)_(n)aconitate hydratase and an iso(homo)_(n)citratedehydrogenase.

In some embodiments, a (homo)_(n)citrate synthase can be classified, forexample, under EC 2.3.3.14 or EC 2.3.3.13, such as the gene product ofaksA from Methanocaldococcus jannaschii (see Genbank Accession No.AAB98494.1).

In some embodiments, the combination of (homo)_(n)citrate dehydrataseand (homo)_(n)aconitate hydratase may be classified, for example, underEC 4.2.1.- (e.g., EC 4.2.1.114, EC 4.2.1.36 or EC 4.2.1.33), such as thegene product of aksD from Methanocaldococcus jannaschii (see, GenbankAccession No. AAB99007.1) or gene product of aksE fromMethanocaldococcus jannaschii (see, Genbank Accession No. AAB99277.1).The gene products of aksD and aksE are subunits of an enzyme classifiedunder EC 4.2.1.114. Homologs in 2-oxoacid chain elongation from thebranch chain amino acid synthesis pathway this is also true, e.g. Thegene products of LeuC and LeuD are subunits of an enzyme classifiedunder EC 4.2.1.33.

In some embodiments, an iso(homo)_(n)citrate dehydrogenase may beclassified, for example, under EC 1.1.1.- such as EC 1.1.1.85, EC1.1.1.87 or EC 1.1.1.286, such as the gene product of aksF fromMethanocaldococcus jannaschii (see, Genbank Accession No. ACA28837.1).

In some embodiments, the product of two or three chain elongationcycles, 2-oxo-suberate, can be decarboxylated by an indolepyruvatedecarboxylase classified, for example, under EC 4.1.1.43 or EC 4.1.1.74such as the indole-3-pyruvate decarboxylase from Salmonella typhimurium(see, for example, Genbank Accession No. CAC48239.1 in which residue 544can be a leucine or an alanine)

2-oxo-suberate also can be decarboxylated by a 2-oxoglutaratedehydrogenase complex comprised of enzymes homologous to enzymesclassified, for example, under EC 1.2.4.2, EC 1.8.1.4, and EC 2.3.1.61.The 2-oxoglutarate dehydrogenase complex contains multiple copies of a2-oxoglutarate dehydrogenase classified, for example, under EC 1.2.4.2bound to a core of dihydrolipoyllysine-residue succinyltransferasesclassified, for example, under EC 2.3.1.61, which also binds multiplecopies of a dihydrolipoyl dehydrogenase classified, for example, underEC 1.8.1.4.

Several analogous 2-oxoacid chain elongation pathway are utilized bymicroorganisms in producing branch chain amino acids, lysine, andCoenzyme B (see, for example, Drevland et al., 2007, J. Bacteriol.,189(12), 4391-4400). Using the chain elongation enzymes for Coenzyme Bbiosynthesis (see, for example, Drevland et al., 2008, J. Biol. Chem.,283(43), 28888-28896; Howell et al., 2000, J. Bacteriol., 182(7),5013-5016), the chain of 2-oxoglutrate can be elongated to the C8dicarboxylic acid, 2-oxosuberate. Similarly, using the chain elongationenzymes associated with branch chain amino acid biosynthesis,2-isopropylmalate synthase encoded by LeuA, can be engineered to acceptlonger chain substrates allowing chain elongation to C7/C8 (Zhang etal., 2008, Proc. Natl. Acad. Sci., 105(52), 20653-20658).

A mutant variant of the indolepyruvate decarboxylase from Salmonellatyphimurium has been engineered successfully to selectively acceptlonger chain length substrates. The L544A mutation of the sequenceprovided in Genbank Accession No. CAC48239.1 allowed for 567 timeshigher selectivity towards the C7 2-oxoacid than towards the C52-oxoacid (see, Xiong et al., 2012, Scientific Reports, 2: 311). The2-oxoglutarate dehydrogenase complex has demonstrated activity for2-oxoglutate and 2-oxoadipate (Bunik et al., 2000, Eur. J. Biochem.,267, 3583-3591).

Enzymes Generating the Terminal Carboxyl Groups in the Biosynthesis ofC7 Building Blocks

As depicted in FIG. 2, a terminal carboxyl group can be enzymaticallyformed using an thioesterase, an aldehyde dehydrogenase, a7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, aCoA-transferase or a reversible CoA-ligase.

In some embodiments, the second terminal carboxyl group leading to thesynthesis of a C7 building block is enzymatically formed by athioesterase classified under EC 3.1.2.-, such as the gene product ofYciA, tesB (Genbank Accession No. AAA24665.1, SEQ ID NO: 1) or Acot13(see, for example, Cantu et al., Protein Science, 2010, 19, 1281-1295;Zhuang et al., Biochemistry, 2008, 47(9), 2789-2796; or Naggert et al.,J. Biol. Chem., 1991, 266(17), 11044-11050).

In some embodiments, the second terminal carboxyl group leading to thesynthesis of pimelic acid is enzymatically formed by an aldehydedehydrogenase classified, for example, under EC 1.2.1.3 (see, forexample, Guerrillot & Vandecasteele, Eur. J. Biochem., 1977, 81,185-192).

In some embodiments, the second terminal carboxyl group leading to thesynthesis of pimelic acid is enzymatically formed by a 6-oxohexanoatedehydrogenase or a 7-oxoheptanoate dehydrogenase classified under EC1.2.1.-, such as the gene product of ChnE from Acinetobacter sp. or ThnGfrom Sphingomonas macrogolitabida (see, for example, Iwaki et al., Appl.Environ. Microbiol., 1999, 65(11), 5158-5162; or López-Sánchez et al.,Appl. Environ. Microbiol., 2010, 76(1), 110-118). For example, a6-oxohexanoate dehydrogenase can be classified under EC 1.2.1.63. Forexample, a 7-oxoheptanoate dehydrogenase can be classified under EC1.2.1.-.

In some embodiments, the second terminal carboxyl group leading to thesynthesis of pimelic acid is enzymatically formed by a CoA-transferasesuch as a glutaconate CoA-transferase classified, for example, under EC2.8.3.12 such as from Acidaminococcus fermentans. See, for example,Buckel et al., 1981, Eur. J. Biochem., 118:315-321.

In some embodiments, the second terminal carboxyl group leading to thesynthesis of pimelic acid is enzymatically formed by a reversibleCoA-ligase such as a succinate-CoA ligase classified, for example, underEC 6.2.1.5 such as from Thermococcus kodakaraensis. See, for example,Shikata et al., 2007, J. Biol. Chem., 282(37):26963-26970.

Enzymes Generating the Terminal Amine Groups in the Biosynthesis of C7Building Blocks

As depicted in FIG. 3 and FIG. 4, terminal amine groups can beenzymatically formed using a ω-transaminase or a deacetylase.

In some embodiments, the first or second terminal amine group leading tothe synthesis of 7-aminoheptanoic acid is enzymatically formed by aω-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19,EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as that obtained fromChromobacterium violaceum (Genbank Accession No. AAQ59697.1, SEQ ID NO:8), Pseudomonas aeruginosa (Genbank Accession No. AAG08191.1, SEQ ID NO:9), Pseudomonas syringae (Genbank Accession No. AAY39893.1, SEQ ID NO:10), Rhodobacter sphaeroides (Genbank Accession No. ABA81135.1, SEQ IDNO: 11), Escherichia coli (Genbank Accession No. AAA57874.1, SEQ ID NO:12), Vibrio Fluvialis (Genbank Accession No. AAA57874.1, SEQ ID NO: 13),Streptomyces griseus, or Clostridium viride. Some of theseω-transaminases are diamine ω-transaminases (e.g., SEQ ID NO:12). Forexample, the ω-transaminases classified, for example, under EC 2.6.1.29or EC 2.6.1.82 may be diamine ω-transaminases.

The reversible ω-transaminase from Chromobacterium violaceum (GenbankAccession No. AAQ59697.1, SEQ ID NO: 8) has demonstrated analogousactivity accepting 6-aminohexanoic acid as amino donor, thus forming thefirst terminal amine group in adipate semialdehyde (Kaulmann et al.,Enzyme and Microbial Technology, 2007, 41, 628-637).

The reversible 4-aminobubyrate:2-oxoglutarate transaminase fromStreptomyces griseus has demonstrated analogous activity for theconversion of 6-aminohexanoate to adipate semialdehyde (Yonaha et al.,Eur. J. Biochem., 1985, 146:101-106).

The reversible 5-aminovalerate transaminase from Clostridium viride hasdemonstrated analogous activity for the conversion of 6-aminohexanoateto adipate semialdehyde (Barker et al., J. Biol. Chem., 1987, 262(19),8994-9003).

In some embodiments, a terminal amine group leading to the synthesis of7-aminoheptanoate or heptamethylenediamine is enzymatically formed by adiamine ω-transaminase. For example, the second terminal amino group canbe enzymatically formed by a diamine ω-transaminase classified, forexample, under EC 2.6.1.29 or classified, for example, under EC2.6.1.82, such as the gene product of YgjG from E. coli (GenbankAccession No. AAA57874.1, SEQ ID NO: 12).

The gene product of ygjG accepts a broad range of diamine carbon chainlength substrates, such as putrescine, cadaverine and spermidine (see,for example, Samsonova et al., BMC Microbiology, 2003, 3:2).

The diamine ω-transaminase from E. coli strain B has demonstratedactivity for 1,7 diaminoheptane (Kim, The Journal of Chemistry, 1964,239(3), 783-786).

In some embodiments, the second terminal amine group leading to thesynthesis of heptamethylenediamine is enzymatically formed by adeacetylase such as acetylputrescine deacetylase classified, forexample, under EC 3.5.1.62. The acetylputrescine deacetylase fromMicrococcus luteus K-11 accepts a broad range of carbon chain lengthsubstrates, such as acetylputrescine, acetylcadaverine andN⁸-acetylspermidine (see, for example, Suzuki et al., 1986, BBA—GeneralSubjects, 882(1):140-142).

Enzymes Generating the Terminal Hydroxyl Groups in the Biosynthesis ofC7 Building Blocks

As depicted in FIG. 5 and FIG. 6, a terminal hydroxyl group can beenzymatically formed using an alcohol dehydrogenase.

In some embodiments, the second terminal hydroxyl group leading to thesynthesis of 1,7 heptanediol is enzymatically formed by an alcoholdehydrogenase classified, for example, under EC 1.1.1.- (e.g., EC1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184).

Biochemical Pathways Pathways Using 2-Oxoadipate as Central Metaboliteto C7 Building Blocks, Pimelate Semialdehyde and Pimeloyl-CoA

In some embodiments, pimeloyl-CoA or pimelate semialdehyde can besynthesized from the central metabolite, 2-oxoglutarate, by three cyclesof 2-oxoacid chain elongation by conversion of 2-oxoglutrate to(Homo)₁citrate by a (Homo)_(n)citrate synthase classified, for example,under EC 2.3.3.14 or EC 2.3.3.13 (see, e.g., AksA, Genbank Accession No.AAB98494.1); followed by conversion to iso(homo)₁citrate by a(homo)_(n)citrate dehydratase and a (homo)_(n)aconitate hydrataseclassified, for example, under EC 4.2.1.114, EC 4.2.1.36 or EC 4.2.1.33(see, e.g., AksD and AksE, Genbank Accession Nos. AAB99007.1 andAAB99277.1); followed by conversion to 2-oxoadipate by aniso(homo)_(n)citrate dehydrogenase classified, for example, under EC1.1.1.85, EC 1.1.1.87 or EC 1.1.1.286 (see, e.g., AksF, GenbankAccession No. AAB98494.1); followed by conversion to (Homo)₂citrate by a(Homo)_(n)citrate synthase classified, for example, under EC 2.3.3.14 orEC 2.3.3.13 (see, e.g., Genbank Accession No. AAB98494.1); followed byconversion to iso(homo)₂citrate (also known as1-hydroxypentane-1,2,5-tricarboxylate or threo-iso(homo)₂citrate) by a(homo)_(n)citrate dehydratase and a (homo)_(n)aconitate hydrataseclassified, for example, under EC 4.2.1.114, EC 4.2.1.36 or EC 4.2.1.33(see, e.g., Genbank Accession Nos. AAB99007.1 and AAB99277.1); followedby conversion to 2-oxo-pimelate by an iso(homo)_(n)citrate dehydrogenaseclassified under, for example, EC 1.1.1.85, EC 1.1.1.87, or EC 1.1.1.286(e.g., Genbank Accession No. AAB98494.1); followed by conversion to(Homo)₃citrate by a (Homo)_(n)citrate synthase classified, for example,under EC 2.3.3.14 or EC 2.3.3.13 (see, e.g., Genbank Accession No.AAB98494.1); followed by conversion to iso(homo)₃citrate by a(homo)_(n)citrate dehydratase and a (homo)_(n)aconitate hydrataseclassified, for example, under EC 4.2.1.114, EC 4.2.1.36 or EC 4.2.1.33(e.g., Genbank Accession Nos. AAB99007.1 and AAB99277.1); followed byconversion to 2-oxo-suberate by an iso(homo)_(n)citrate dehydrogenaseclassified under, for example, EC 1.1.1.85, EC 1.1.1.87, or EC 1.1.1.286(e.g., Genbank Accession No. AAB98494.1). Pimeloyl-CoA can be producedby conversion of 2-oxo-suberate to pimeloyl-CoA by a 2-oxoglutaratedehydrogenase complex containing enzymes classified, for example, underEC 1.2.4.2, EC 1.8.1.4 and EC 2.3.1.61. Pimelate semialdehyde can beproduced by conversion of 2-oxo-suberate to pimelate semialdehyde by adecarboxylase classified, for example, EC 4.1.1.- (e.g., EC 4.1.1.43 andEC 4.1.1.74) (see e.g., Genbank Accession No. CAC48239.1). See, FIG. 1.

In some embodiments, pimelate semialdehyde or pimeloyl-CoA can besynthesized from the central metabolite, 2-oxoadipate, as described inFIG. 1.

Pathways Using Pimeloyl-CoA or Pimelate Semialdehyde as CentralPrecursors to Pimelate

In some embodiments, pimelic acid is synthesized from the centralprecursor, pimeloyl-CoA, by conversion of pimeloyl-CoA to pimelatesemialdehyde by an acetylating aldehyde dehydrogenase classified, forexample, under EC 1.2.1.10 such as the gene product of PduB or PduP(see, for example, Lan et al., 2013, Energy Environ. Sci., 6:2672-2681);followed by conversion to pimelic acid by a 7-oxoheptanoatedehydrogenase classified, for example, under EC 1.2.1.- such as the geneproduct of ThnG, a 6-oxohexanoate dehydrogenase classified, for example,under EC 1.2.1.- such as the gene product of ChnE, or an aldehydedehydrogenase (classified, for example, under C 1.2.1.3). See FIG. 2.

In some embodiments, pimelic acid is synthesized from the centralprecursor, pimeloyl-CoA, by conversion of pimeloyl-CoA to pimelate by athioesterase classified, for example, under EC 3.1.2.- such as the geneproducts of YciA, tesB (Genbank Accession No. AAA24665.1, SEQ ID NO: 1)or Acot13. See FIG. 2.

In some embodiments, pimelate is synthesized from the central precursor,pimeloyl-CoA, by conversion of pimeloyl-CoA to pimelate by aCoA-transferase such as a glutaconate CoA-transferase classified, forexample, under EC 2.8.3.12. See FIG. 2. In some embodiments, pimelate issynthesized from the central precursor, pimeloyl-CoA, by conversion ofpimeloyl-CoA to pimelate by a reversible CoA-ligase such as a reversiblesuccinate CoA-ligase classified, for example, under EC 6.2.1.5. See FIG.2.

In some embodiments, pimelate is synthesized from the central precursor,pimelate semialdehyde, by conversion of pimelate semialdehyde topimelate by a 6-oxohexanoate dehydrogenase or a 7-oxoheptanoatedehydrogenase (classified, for example, under EC 1.2.1.-) such as thegene product of ThnG or ChnE, or an aldehyde dehydrogenase (classified,for example, under EC 1.2.1.3). See FIG. 2.

Pathways Using Pimeloyl-CoA or Pimelate Semialdehyde as CentralPrecursor to 7-Aminoheptanoate

In some embodiments, 7-aminoheptanoate is synthesized from the centralprecursor, pimeloyl-CoA, by conversion of pimeloyl-CoA to pimelatesemialdehyde by an acetylating aldehyde dehydrogenase classified, forexample, EC 1.2.1.10, such as the gene product of PduB or PduP; followedby conversion of pimelate semialdehyde to 7-aminoheptanoate by aω-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19,EC 2.6.1.29, EC 2.6.1.48 or EC 2.6.1.82. See FIG. 3.

In some embodiments, 7-aminoheptanoate is synthesized from the centralprecursor, pimelate semialdehyde, by conversion of pimelate semialdehydeto 7-aminoheptanoate by a ω-transaminase (e.g., EC 2.6.1.18, EC2.6.1.19, or EC 2.6.1.48). See FIG. 3.

In some embodiments, 7-aminoheptanoate is synthesized from the centralprecursor, pimelate, by conversion of pimelate to pimelate semialdehydeby a carboxylate reductase classified, for example, under EC 1.2.99.6such as the gene product of car in combination with a phosphopantetheinetransferase enhancer (e.g., encoded by a sfp (Genbank Accession No.CAA44858.1, SEQ ID NO:14) gene from Bacillus subtilis or npt (GenbankAccession No. ABI83656.1, SEQ ID NO:15) gene from Nocardia) or the geneproducts of GriC and GriD from Streptomyces griseus; followed byconversion of pimelate semialdehyde to 7-aminoheptanoate by aω-transaminase (e.g., EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.48, EC2.6.1.29, EC 2.6.1.82 such as SEQ ID NOs:8-13). The carboxylatereductase can be obtained, for example, from Mycobacterium marinum(Genbank Accession No. ACC40567.1, SEQ ID NO: 2), Mycobacteriumsmegmatis (Genbank Accession No. ABK71854.1, SEQ ID NO: 3), Segniliparusrugosus (Genbank Accession No. EFV11917.1, SEQ ID NO: 4), Mycobacteriumsmegmatis (Genbank Accession No. ABK75684.1, SEQ ID NO: 5),Mycobacterium massiliense (Genbank Accession No. EIV11143.1, SEQ ID NO:6), or Segniliparus rotundus (Genbank Accession No. ADG98140.1, SEQ IDNO: 7). See FIG. 3.

Pathway Using 7-Aminoheptanoate, 7-Hydroxyheptanoate or PimelateSemialdehyde as Central Precursor to Heptamethylenediamine

In some embodiments, heptamethylenediamine is synthesized from thecentral precursor, 7-aminoheptanoate, by conversion of 7-aminoheptanoateto 7-aminoheptanal by a carboxylate reductase classified, for example,under EC 1.2.99.6 such as the gene product of car (see above) incombination with a phosphopantetheine transferase enhancer (e.g.,encoded by a sfp (Genbank Accession No. CAA44858.1, SEQ ID NO:14) genefrom Bacillus subtilis or npt (Genbank Accession No. ABI83656.1, SEQ IDNO:15) gene from Nocardia) or the gene product of GriC & GriD (Suzuki etal., J. Antibiot., 2007, 60(6), 380-387); followed by conversion of7-aminoheptanal to heptamethylenediamine by a ω-transaminase (e.g.,classified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC2.6.1.48, or EC 2.6.1.82 such as SEQ ID NOs:8-13, see above). See FIG.4.

The carboxylate reductase encoded by the gene product of car and thephosphopantetheine transferase enhancer npt or sfp has broad substratespecificity, including terminal difunctional C4 and C5 carboxylic acids(Venkitasubramanian et al., Enzyme and Microbial Technology, 2008, 42,130-137).

In some embodiments, heptamethylenediamine is synthesized from thecentral precursor, 7-hydroxyheptanoate (which can be produced asdescribed in FIG. 5), by conversion of 7-hydroxyheptanoate to7-hydroxyheptanal by a carboxylate reductase classified, for example,under EC 1.2.99.6 such as the gene product of car (see above) incombination with a phosphopantetheine transferase enhancer (e.g.,encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia)or the gene product of GriC & GriD (Suzuki et al., J. Antibiot., 2007,60(6), 380-387); followed by conversion of 7-aminoheptanal to7-aminoheptanol by a ω-transaminase classified, for example, under EC2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such asSEQ ID NOs:8-13, see above; followed by conversion to 7-aminoheptanal byan alcohol dehydrogenase classified, for example, under EC 1.1.1.-(e.g., EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) such as thegene product of YMR318C (classified, for example, under EC 1.1.1.2, seeGenbank Accession No. CAA90836.1) or YqhD (from E. coli, GenBankAccession No. AAA69178.1) (Liu et al., Microbiology, 2009, 155,2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172; Jarboe,2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or the proteinhaving GenBank Accession No. CAA81612.1 (from Geobacillusstearothermophilus); followed by conversion to heptamethylenediamine bya ω-transaminase classified, for example, under EC 2.6.1.18, EC2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as SEQ IDNOs:8-13, see above. See FIG. 4.

In some embodiments, heptamethylenediamine is synthesized from thecentral precursor, 7-aminoheptanoate, by conversion of 7-aminoheptanoateto N7-acetyl-7-aminoheptanoate by a N-acetyltransferase such as a lysineN-acetyltransferase classified, for example, under EC 2.3.1.32; followedby conversion to N7-acetyl-7-aminoheptanal by a carboxylate reductaseclassified, for example, under EC 1.2.99.6 such as the gene product ofcar (see above) in combination with a phosphopantetheine transferaseenhancer (e.g., encoded by a sfp gene from Bacillus subtilis or npt genefrom Nocardia) or the gene product of GriC & GriD; followed byconversion to N7-acetyl-1,7-diaminoheptane by a ω-transaminaseclassified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC2.6.1.48, EC 2.6.1.46, or EC 2.6.1.82 such as SEQ ID NOs:8-13, seeabove; followed by conversion to heptamethylenediamine by anacetylputrescine deacylase classified, for example, under EC 3.5.1.62.See, FIG. 4.

In some embodiments, heptamethylenediamine is synthesized from thecentral precursor, pimelate semialdehyde, by conversion of pimelatesemialdehyde to heptanedial by a carboxylate reductase classified, forexample, under EC 1.2.99.6 such as the gene product of car (see above)in combination with a phosphopantetheine transferase enhancer (e.g.,encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia)or the gene product of GriC & GriD; followed by conversion to7-aminoheptanal by a ω-transaminase classified, for example, under EC2.6.1.18, EC 2.6.1.19, or EC 2.6.1.48; followed by conversion toheptamethylenediamine by a ω-transaminase classified, for example, underEC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, EC 2.6.1.46, or EC2.6.1.82 such as SEQ ID NOs:8-13, see above. See FIG. 4.

Pathways Using Pimelate or Pimelate Semialdehyde as Central Precursor to1,7-Heptanediol

In some embodiments, 7-hydroxyheptanoate is synthesized from the centralprecursor, pimelate, by conversion of pimelate to pimelate semialdehydeby a carboxylate reductase classified, for example, under EC 1.2.99.6such as the gene product of car (see above) in combination with aphosphopantetheine transferase enhancer (e.g., encoded by a sfp genefrom Bacillus subtilis or npt gene from Nocardia) or the gene product ofGriC & GriD; followed by conversion to 7-hydroxyheptanoate by adehydrogenase classified, for example, under EC 1.1.1.- such as a6-hydroxyhexanoate dehydrogenase classified, for example, under EC1.1.1.258 such as the gene from of ChnD or a 5-hydroxypentanoatedehydrogenase classified, for example, under EC 1.1.1.- such as the geneproduct of CpnD (see, for example, Iwaki et al., 2002, Appl. Environ.Microbiol., 68(11):5671-5684) or a 4-hydroxybutyrate dehydrogenase suchas gabD (see, for example, Liitke-Eversloh & Steinbiichel, 1999, FEMSMicrobiology Letters, 181(1):63-71). See FIG. 5. Pimelate semialdehydealso can be produced from pimeloyl-CoA using an acetylating aldehydedehydrogenase as described above. See, also FIG. 5.

In some embodiments, 1,7 heptanediol is synthesized from the centralprecursor, 7-hydroxyheptanoate, by conversion of 7-hydroxyheptanoate to7-hydroxyheptanal by a carboxylate reductase classified, for example,under EC 1.2.99.6 such as the gene product of car (see above) incombination with a phosphopantetheine transferase enhancer (e.g.,encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia)or the gene product of GriC & GriD; followed by conversion of7-hydroxyheptanal to 1,7 heptanediol by an alcohol dehydrogenaseclassified, for example, under EC 1.1.1.- such as EC 1.1.1.1, EC1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) such as the gene product ofYMR318C or YqhD (see, e.g., Liu et al., Microbiology, 2009, 155,2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172; orJarboe, 2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or theprotein having GenBank Accession No. CAA81612.1 (from Geobacillusstearothermophilus). See, FIG. 6.

Cultivation Strategy

In some embodiments, the cultivation strategy entails achieving anaerobic, anaerobic, micro-aerobic, or mixed oxygen/denitrificationcultivation condition. Enzymes characterized in vitro as being oxygensensitive require a micro-aerobic cultivation strategy maintaining avery low dissolved oxygen concentration (See, for example, Chayabatra &Lu-Kwang, Appl. Environ. Microbiol., 2000, 66(2), 493 0 498; Wilson andBouwer, 1997, Journal of Industrial Microbiology and Biotechnology,18(2-3), 116-130).

In some embodiments, the cultivation strategy entails nutrientlimitation such as nitrogen, phosphate or oxygen limitation.

In some embodiments, a final electron acceptor other than oxygen such asnitrates can be utilized.

In some embodiments, a cell retention strategy using, for example,ceramic hollow fiber membranes can be employed to achieve and maintain ahigh cell density during either fed-batch or continuous fermentation.

In some embodiments, the principal carbon source fed to the fermentationin the synthesis of one or more C7 building blocks can derive frombiological or non-biological feedstocks.

In some embodiments, the biological feedstock can be, can include, orcan derive from, monosaccharides, disaccharides, lignocellulose,hemicellulose, cellulose, lignin, levulinic acid and formic acid,triglycerides, glycerol, fatty acids, agricultural waste, condenseddistillers' solubles, or municipal waste.

The efficient catabolism of crude glycerol stemming from the productionof biodiesel has been demonstrated in several microorganisms such asEscherichia coli, Cupriavidus necator, Pseudomonas oleavorans,Pseudomonas putida and Yarrowia lipolytica (Lee et al., Appl. Biochem.Biotechnol., 2012, 166, 1801-1813; Yang et al., Biotechnology forBiofuels, 2012, 5:13; Meijnen et al., Appl. Microbiol. Biotechnol.,2011, 90, 885-893).

The efficient catabolism of lignocellulosic-derived levulinic acid hasbeen demonstrated in several organisms such as Cupriavidus necator andPseudomonas putida in the synthesis of 3-hydroxyvalerate via theprecursor propanoyl-CoA (Jaremko and Yu, Journal of Biotechnology, 2011,155, 2011, 293-298; Martin and Prather, Journal of Biotechnology, 2009,139, 61-67).

The efficient catabolism of lignin-derived aromatic compounds such asbenzoate analogues has been demonstrated in several microorganisms suchas Pseudomonas putida, Cupriavidus necator (Bugg et al., Current Opinionin Biotechnology, 2011, 22, 394-400; Perez-Pantoja et al., FEMSMicrobiol. Rev., 2008, 32, 736-794).

The efficient utilization of agricultural waste, such as olive millwaste water has been demonstrated in several microorganisms, includingYarrowia lipolytica (Papanikolaou et al., Bioresour. Technol., 2008,99(7), 2419-2428).

The efficient utilization of fermentable sugars such as monosaccharidesand disaccharides derived from cellulosic, hemicellulosic, cane and beetmolasses, cassava, corn and other agricultural sources has beendemonstrated for several microorganism such as Escherichia coli,Corynebacterium glutamicum and Lactobacillus delbrueckii and Lactococcuslactis (see, e.g., Hermann et al, Journal of Biotechnology, 2003, 104,155-172; Wee et al., Food Technol. Biotechnol., 2006, 44(2), 163-172;Ohashi et al., Journal of Bioscience and Bioengineering, 1999, 87(5),647-654).

The efficient utilization of furfural, derived from a variety ofagricultural lignocellulosic sources, has been demonstrated forCupriavidus necator (Li et al., Biodegradation, 2011, 22, 1215-1225).

In some embodiments, the non-biological feedstock can be or can derivefrom natural gas, syngas, CO₂/H₂, methanol, ethanol, benzoic acid,non-volatile residue (NVR), a caustic wash waste stream from cyclohexaneoxidation processes, or terephthalic acid/isophthalic acid mixture wastestreams.

The efficient catabolism of methanol has been demonstrated for themethylotrophic yeast Pichia pastoris.

The efficient catabolism of ethanol has been demonstrated forClostridium kluyveri (Seedorf et al., Proc. Natl. Acad. Sci. USA, 2008,105(6) 2128-2133).

The efficient catabolism of CO₂ and H₂, which may be derived fromnatural gas and other chemical and petrochemical sources, has beendemonstrated for Cupriavidus necator (Prybylski et al., Energy,Sustainability and Society, 2012, 2:11).

The efficient catabolism of syngas has been demonstrated for numerousmicroorganisms, such as Clostridium ljungdahlii and Clostridiumautoethanogenum (Kopke et al., Applied and Environmental Microbiology,2011, 77(15), 5467-5475).

The efficient catabolism of the non-volatile residue waste stream fromcyclohexane processes has been demonstrated for numerous microorganisms,such as Delftia acidovorans and Cupriavidus necator (Ramsay et al.,Applied and Environmental Microbiology, 1986, 52(1), 152-156). In someembodiments, the host microorganism is a prokaryote. For example, theprokaryote can be a bacterium from the genus Escherichia such asEscherichia coli; from the genus Clostridia such as Clostridiumljungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; fromthe genus Corynebacteria such as Corynebacterium glutamicum; from thegenus Cupriavidus such as Cupriavidus necator or Cupriavidusmetallidurans; from the genus Pseudomonas such as Pseudomonasfluorescens, Pseudomonas putida or Pseudomonas oleavorans; from thegenus Delftia such as Delftia acidovorans; from the genus Bacillus suchas Bacillus subtillis; from the genus Lactobacillus such asLactobacillus delbrueckii; or from the genus Lactococcus such asLactococcus lactis. Such prokaryotes also can be a source of genes toconstruct recombinant host cells described herein that are capable ofproducing one or more C7 building blocks.

In some embodiments, the host microorganism is a eukaryote. For example,the eukaryote can be a filamentous fungus, e.g., one from the genusAspergillus such as Aspergillus niger. Alternatively, the eukaryote canbe a yeast, e.g., one from the genus Saccharomyces such as Saccharomycescerevisiae; from the genus Pichia such as Pichia pastoris; or from thegenus Yarrowia such as Yarrowia lipolytica; from the genus Issatchenkiasuch as Issathenkia orientalis; from the genus Debaryomyces such asDebaryomyces hansenii; from the genus Arxula such as Arxulaadenoinivorans; or from the genus Kluyveromyces such as Kluyveromyceslactis. Such eukaryotes also can be a source of genes to constructrecombinant host cells described herein that are capable of producingone or more C7 building blocks.

Metabolic Engineering

The present document provides methods involving less than all the stepsdescribed for all the above pathways. Such methods can involve, forexample, one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve or more of such steps. Where less than all the steps areincluded in such a method, the first, and in some embodiments the only,step can be any one of the steps listed.

Furthermore, recombinant hosts described herein can include anycombination of the above enzymes such that one or more of the steps,e.g., one, two, three, four, five, six, seven, eight, nine, ten, or moreof such steps, can be performed within a recombinant host. This documentprovides host cells of any of the genera and species listed andgenetically engineered to express one or more (e.g., two, three, four,five, six, seven, eight, nine, 10, 11, 12 or more) recombinant forms ofany of the enzymes recited in the document. Thus, for example, the hostcells can contain exogenous nucleic acids encoding enzymes catalyzingone or more of the steps of any of the pathways described herein.

In addition, this document recognizes that where enzymes have beendescribed as accepting CoA-activated substrates, analogous enzymeactivities associated with [acp]-bound substrates exist that are notnecessarily in the same enzyme class.

Also, this document recognizes that where enzymes have been describedaccepting (R)-enantiomers of substrate, analogous enzyme activitiesassociated with (S)-enantiomer substrates exist that are not necessarilyin the same enzyme class.

This document also recognizes that where an enzyme is shown to accept aparticular co-factor, such as NADPH, or a co-substrate, such asacetyl-CoA, many enzymes are promiscuous in terms of accepting a numberof different co-factors or co-substrates in catalyzing a particularenzyme activity. Also, this document recognizes that where enzymes havehigh specificity for e.g., a particular co-factor such as NADH, anenzyme with similar or identical activity that has high specificity forthe co-factor NADPH may be in a different enzyme class.

In some embodiments, the enzymes in the pathways outlined herein are theresult of enzyme engineering via non-direct or rational enzyme designapproaches with aims of improving activity, improving specificity,reducing feedback inhibition, reducing repression, improving enzymesolubility, changing stereo-specificity, or changing co-factorspecificity.

In some embodiments, the enzymes in the pathways outlined herein can begene dosed (i.e., overexpressed by having a plurality of copies of thegene in the host organism), into the resulting genetically modifiedorganism via episomal or chromosomal integration approaches.

In some embodiments, genome-scale system biology techniques such as FluxBalance Analysis can be utilized to devise genome scale attenuation orknockout strategies for directing carbon flux to a C7 building block.

Attenuation strategies include, but are not limited to; the use oftransposons, homologous recombination (double cross-over approach),mutagenesis, enzyme inhibitors and RNA interference (RNAi).

In some embodiments, fluxomic, metabolomic and transcriptomal data canbe utilized to inform or support genome-scale system biology techniques,thereby devising genome scale attenuation or knockout strategies indirecting carbon flux to a C7 building block.

In some embodiments, the host microorganism's tolerance to highconcentrations of a C7 building block can be improved through continuouscultivation in a selective environment.

In some embodiments, the host microorganism's endogenous biochemicalnetwork can be attenuated or augmented to ensure the intracellularavailability of 2-oxoglutarate (2) create an NAD⁺ imbalance that mayonly be balanced via the formation of a C7 building block, (3) preventdegradation of central metabolites or central precursors leading to andincluding C7 building blocks and (4) ensure efficient efflux from thecell.

In some embodiments requiring the intracellular availability of2-oxoglutarate, a PEP carboxykinase or PEP carboxylase can beoverexpressed in the host to generate anaplerotic carbon flux into theKrebs cycle towards 2-oxoglutarate (Schwartz et al., 2009, Proteomics,9, 5132-5142).

In some embodiments requiring the intracellular availability of2-oxoglutarate, a pyruvate carboxylase can be overexpressed in the hostto generated anaplerotic carbon flux into the Krebs cycle towards2-oxoglutarate (Schwartz et al., 2009, Proteomics, 9, 5132-5142).

In some embodiments requiring the intracellular availability of2-oxoglutarate, a PEP synthase can be overexpressed in the host toenhance the flux from pyruvate to PEP, thus increasing the carbon fluxinto the Krebs cycle via PEP carboxykinase or PEP carboxylase (Schwartzet al., 2009, Proteomics, 9, 5132-5142).

In some embodiments where the host microorganism uses the lysinebiosynthesis pathway via meso-2,6-diaminopimelate, the genes encodingthe synthesis of 2-oxoadipate from 2-oxoglutarate are gene dosed intothe host.

In some embodiments where the host microorganism uses the lysinebiosynthesis pathway via 2-oxoadipate, the genes encoding the synthesisof lysine via meso-2,6-diaminopimelate are gene dosed into the host.

In some embodiments preventing the degradation of NADH formed during thesynthesis of C7 building blocks to by-products, an endogenous geneencoding an enzyme, such as lactate dehydrogenase, that catalyzes thedegradation of pyruvate to lactate such as ldhA is attenuated (Shen etal., Appl. Environ. Microbiol., 2011, 77(9), 2905-2915).

In some embodiments preventing the degradation of NADH formed during thesynthesis of C7 building blocks to by-products, an endogenous gene, suchas menaquinol-fumarate oxidoreductase, encoding an enzyme that catalyzesthe degradation of phophoenolpyruvate to succinate such as frdBC isattenuated (see, e.g., Shen et al., 2011, supra).

In some embodiments preventing the degradation of NADH to by-productsformed during the synthesis of C7 building blocks, an endogenous geneencoding an enzyme that catalyzes the degradation of acetyl-CoA toethanol such as the alcohol dehydrogenase encoded by adhE is attenuated(Shen et al., 2011, supra).

In some embodiments, an endogenous gene encoding an enzyme thatcatalyzes the degradation of pyruvate to ethanol such as pyruvatedecarboxylase is attenuated.

In some embodiments, an endogenous gene encoding an enzyme thatcatalyzes the generation of isobutanol such as a 2-oxoacid decarboxylasecan be attenuated.

In some embodiments, where a pathway requires excess NAD⁺ co-factor forC7 building block synthesis, a gene encoding aformate dehydrogenase canbe attenuated in the host organism (Shen et al., 2011, supra).

In some embodiments, endogenous enzymes facilitating the conversion ofNADPH to NADH are attenuated, such as a NADPH-specific glutamatedehydrogenases such as classified, for example, under EC 1.4.1.4.

In some embodiments, transhydrogenases classified, for example, under EC1.6.1.1, EC 1.6.1.2 or EC 1.6.1.3, may be attenuated.

In some embodiments, an endogenous gene encoding a glutamatedehydrogenase (EC 1.4.1.3) that utilizes both NADH and NADPH asco-factors is attenuated.

In some embodiments using hosts that naturally accumulatepolyhydroxyalkanoates, an endogenous gene encoding a polymer synthaseenzyme can be attenuated in the host strain.

In some embodiments, a L-alanine dehydrogenase can be overexpressed inthe host to regenerate L-alanine from pyruvate as amino donor forω-transaminase reactions.

In some embodiments, a NADH-specific L-glutamate dehydrogenase can beoverexpressed in the host to regenerate L-glutamate from 2-oxoglutarateas amino donor for ω-transaminase reactions.

In some embodiments, enzymes such as pimeloyl-CoA dehydrogenaseclassified under, for example, EC 1.3.1.62; am acyl-CoA dehydrogenaseclassified under, for example, EC 1.3.8.7 or EC 1.3.8.1; and/or aglutaryl-CoA dehydrogenase classified under, for example, EC 1.3.8.6that degrade central metabolites and central precursors leading to andincluding C7 building blocks can be attenuated.

In some embodiments, endogenous enzymes activating C7 building blocksvia Coenzyme A esterification such as CoA-ligases such as pimeloyl-CoAsynthetase classified under, for example, EC 6.2.1.14 can be attenuated.

In some embodiments, the efflux of a C7 building block across the cellmembrane to the extracellular media can be enhanced or amplified bygenetically engineering structural modifications to the cell membrane orincreasing any associated transporter activity for a C7 building block.

The efflux of heptamethylenediamine can be enhanced or amplified byoverexpressing broad substrate range multidrug transporters such as Bltfrom Bacillus subtilis (Woolridge et al., 1997, J. Biol. Chem.,272(14):8864-8866); AcrB and AcrD from Escherichia coli (Elkins &Nikaido, 2002, J. Bacteriol., 184(23), 6490-6499) or NorA fromStaphylococcus aereus (Ng et al., 1994, Antimicrob Agents Chemother,38(6), 1345-1355) or Bmr from Bacillus subtilis (Neyfakh, 1992,Antimicrob Agents Chemother, 36(2), 484-485).

The efflux of 7-aminoheptanoate and heptamethylenediamine can beenhanced or amplified by overexpressing the solute transporters such asthe lysE transporter from Corynebacterium glutamicum (Bellmann et al.,2001, Microbiology, 147, 1765-1774).

The efflux of pimelic acid can be enhanced or amplified byoverexpressing a dicarboxylate transporter such as the SucE transporterfrom Corynebacterium glutamicum (Huhn et al., Appl. Microbiol. &Biotech., 89(2), 327-335).

Producing C7 Building Blocks Using a Recombinant Host

Typically, one or more C7 building blocks can be produced by providing ahost microorganism and culturing the provided microorganism with aculture medium containing a suitable carbon source as described above.In general, the culture media and/or culture conditions can be such thatthe microorganisms grow to an adequate density and produce a C7 buildingblock efficiently. For large-scale production processes, any method canbe used such as those described elsewhere (Manual of IndustrialMicrobiology and Biotechnology, 2^(nd) Edition, Editors: A. L. Demainand J. E. Davies, ASM Press; and Principles of Fermentation Technology,P. F. Stanbury and A. Whitaker, Pergamon). Briefly, a large tank (e.g.,a 100 gallon, 200 gallon, 500 gallon, or more tank) containing anappropriate culture medium is inoculated with a particularmicroorganism. After inoculation, the microorganism is incubated toallow biomass to be produced. Once a desired biomass is reached, thebroth containing the microorganisms can be transferred to a second tank.This second tank can be any size. For example, the second tank can belarger, smaller, or the same size as the first tank. Typically, thesecond tank is larger than the first such that additional culture mediumcan be added to the broth from the first tank. In addition, the culturemedium within this second tank can be the same as, or different from,that used in the first tank.

Once transferred, the microorganisms can be incubated to allow for theproduction of a C7 building block. Once produced, any method can be usedto isolate C7 building blocks. For example, C7 building blocks can berecovered selectively from the fermentation broth via adsorptionprocesses. In the case of pimelic acid and 7-aminoheptanoic acid, theresulting eluate can be further concentrated via evaporation,crystallized via evaporative and/or cooling crystallization, and thecrystals recovered via centrifugation. In the case ofheptamethylenediamine and 1,7-heptanediol, distillation may be employedto achieve the desired product purity.

The invention is further described in the following example, which doesnot limit the scope of the invention described in the claims.

EXAMPLES Example 1 Enzyme Activity of Thioesterases Using Pimeloyl-CoAas a Substrate and Forming Pimelic Acid

A sequence encoding an N-terminal His tag was added to the tesB genefrom Escherichia coli that encodes a thioesterase (SEQ ID NO 1, see FIG.7), such that an N-terminal HIS tagged thioesterase could be produced.The modified tesB gene was cloned into a pET15b expression vector undercontrol of the T7 promoter. The expression vector was transformed into aBL21[DE3] E. coli host. The resulting recombinant E. coli strain wascultivated at 37° C. in a 500 mL shake flask culture containing 50 mLLuria Broth (LB) media and antibiotic selection pressure, with shakingat 230 rpm. The culture was induced overnight at 17° C. using 0.5 mMIPTG.

The pellet from the induced shake flask culture was harvested viacentrifugation. The pellet was resuspended and lysed in Y-per™ solution(ThermoScientific, Rockford, Ill.). The cell debris was separated fromthe supernatant via centrifugation. The thioesterase was purified fromthe supernatant using Ni-affinity chromatography and the eluate wasbuffer exchanged and concentrated via ultrafiltration.

The enzyme activity assay was performed in triplicate in a buffercomposed of 50 mM phosphate buffer (pH=7.4), 0.1 mM Ellman's reagent,and 667 μM of pimeloyl-CoA (as substrate). The enzyme activity assayreaction was initiated by adding 0.8 μM of the tesB gene product to theassay buffer containing the pimeloyl-CoA and incubating at 37° C. for 20min. The release of Coenzyme A was monitored by absorbance at 412 nm.The absorbance associated with the substrate only control, whichcontained boiled enzyme, was subtracted from the active enzyme assayabsorbance and compared to the empty vector control. The gene product oftesB accepted pimeloyl-CoA as substrate as confirmed via relativespectrophotometry (see FIG. 8) and synthesized pimelate as a reactionproduct.

Example 2 Enzyme Activity of ω-Transaminase Using Pimelate Semialdehydeas Substrate and Forming 7-Aminoheptanoate

A sequence encoding an N-terminal His-tag was added to the genes fromChromobacterium violaceum, Pseudomonas syringae, Rhodobactersphaeroides, and Vibrio Fluvialis encoding the ω-transaminases of SEQ IDNOs: 8, 10, 11 and 13, respectively (see FIG. 7) such that N-terminalHIS tagged ω-transaminases could be produced. Each of the resultingmodified genes was cloned into a pET21a expression vector under controlof the T7 promoter and each expression vector was transformed into aBL21[DE3] E. coli host. The resulting recombinant E. coli strains werecultivated at 37° C. in a 250 mL shake flask culture containing 50 mL LBmedia and antibiotic selection pressure, with shaking at 230 rpm. Eachculture was induced overnight at 16° C. using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e., 7-aminoheptanoateto pimelate semialdehyde) were performed in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 10 mM 7-aminoheptanoate,10 mM pyruvate and 100 μM pyridoxyl 5′ phosphate. Each enzyme activityassay reaction was initiated by adding cell free extract of theω-transaminase gene product or the empty vector control to the assaybuffer containing the 7-aminoheptanoate and incubated at 25° C. for 4 h,with shaking at 250 rpm. The formation of L-alanine from pyruvate wasquantified via RP-HPLC.

Each enzyme only control without 7-aminoheptanoate demonstrated low baseline conversion of pyruvate to L-alanine. See FIG. 14. The gene productof SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 13 accepted7-aminoheptanote as substrate as confirmed against the empty vectorcontrol. See FIG. 15.

Enzyme activity in the forward direction (i.e., pimelate semialdehyde to7-aminoheptanoate) was confirmed for the transaminases of SEQ ID NO 10,SEQ ID NO 11 and SEQ ID NO 13. Enzyme activity assays were performed ina buffer composed of a final concentration of 50 mM HEPES buffer(pH=7.5), 10 mM pimelate semialdehyde, 10 mM L-alanine and 100 μMpyridoxyl 5′ phosphate. Each enzyme activity assay reaction wasinitiated by adding a cell free extract of the ω-transaminase geneproduct or the empty vector control to the assay buffer containing thepimelate semialdehyde and incubated at 25° C. for 4 h, with shaking at250 rpm. The formation of pyruvate was quantified via RP-HPLC.

The gene product of SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 13 acceptedpimelate semialdehyde as substrate as confirmed against the empty vectorcontrol. See FIG. 16. The reversibility of the ω-transaminase activitywas confirmed, demonstrating that the ω-transaminases of SEQ ID NO 8,SEQ ID NO 10, SEQ ID NO 11, and SEQ ID NO 13 accepted pimelatesemialdehyde as substrate and synthesized 7-aminoheptanoate as areaction product.

Example 3 Enzyme Activity of Carboxylate Reductase Using Pimelate asSubstrate and Forming Pimelate Semialdehyde

A sequence encoding a HIS-tag was added to the genes from Segniliparusrugosus and Segniliparus rotundus that encode the carboxylate reductasesof SEQ ID NOs: 4 and 7, respectively (see FIG. 7), such that N-terminalHIS tagged carboxylate reductases could be produced. Each of themodified genes was cloned into a pET Duet expression vector along with asfp gene encoding a HIS-tagged phosphopantetheine transferase fromBacillus subtilis, both under the T7 promoter. Each expression vectorwas transformed into a BL21[DE3] E. coli host and the resultingrecombinant E. coli strains were cultivated at 37° C. in a 250 mL shakeflask culture containing 50 mL LB media and antibiotic selectionpressure, with shaking at 230 rpm. Each culture was induced overnight at37° C. using an auto-induction media.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication,and the cell debris was separated from the supernatant viacentrifugation. The carboxylate reductases and phosphopantetheinetransferases were purified from the supernatant using Ni-affinitychromatography, diluted 10-fold into 50 mM HEPES buffer (pH=7.5), andconcentrated via ultrafiltration.

Enzyme activity assays (i.e., from pimelate to pimelate semialdehyde)were performed in triplicate in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 2 mM pimelate, 10 mMMgCl₂, 1 mM ATP and 1 mM NADPH. Each enzyme activity assay reaction wasinitiated by adding purified carboxylate reductase andphosphopantetheine transferase gene products or the empty vector controlto the assay buffer containing the pimelate and then incubated at roomtemperature for 20 min. The consumption of NADPH was monitored byabsorbance at 340 nm. Each enzyme only control without pimelatedemonstrated low base line consumption of NADPH. See FIG. 9.

The gene products of SEQ ID NO 4 and SEQ ID NO 7, enhanced by the geneproduct of sfp, accepted pimelate as substrate, as confirmed against theempty vector control (see FIG. 10), and synthesized pimelatesemialdehyde.

Example 4 Enzyme Activity of Carboxylate Reductase Using7-Hydroxyheptanoate as Substrate and Forming 7-Hydroxyheptanal

A sequence encoding a His-tag was added to the genes from Mycobacteriummarinum, Mycobacterium smegmatis, Segniliparus rugosus, Mycobacteriumsmegmatis, Mycobacterium massiliense, and Segniliparus rotundus thatencode the carboxylate reductases of SEQ ID NOs: 2-7—respectively (seeFIG. 7) such that N-terminal HIS tagged carboxylate reductases could beproduced. Each of the modified genes was cloned into a pET Duetexpression vector alongside a sfp gene encoding a His-taggedphosphopantetheine transferase from Bacillus subtilis, both undercontrol of the T7 promoter.

Each expression vector was transformed into a BL21[DE3] E. coli host andthe resulting recombinant E. coli strains were cultivated at 37° C. in a250 mL shake flask culture containing 50 mL LB media and antibioticselection pressure, with shaking at 230 rpm. Each culture was inducedovernight at 37° C. using an auto-induction media.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugation.The carboxylate reductases and phosphopantetheine transferase werepurified from the supernatant using Ni-affinity chromatography, diluted10-fold into 50 mM HEPES buffer (pH=7.5) and concentrated viaultrafiltration.

Enzyme activity (i.e., 7-hydroxyheptanoate to 7-hydroxyheptanal) assayswere performed in triplicate in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 2 mM 7-hydroxyheptanal, 10mM MgCl₂, 1 mM ATP, and 1 mM NADPH. Each enzyme activity assay reactionwas initiated by adding purified carboxylate reductase andphosphopantetheine transferase or the empty vector control to the assaybuffer containing the 7-hydroxyheptanoate and then incubated at roomtemperature for 20 min. The consumption of NADPH was monitored byabsorbance at 340 nm. Each enzyme only control without7-hydroxyheptanoate demonstrated low base line consumption of NADPH. SeeFIG. 9.

The gene products of SEQ ID NO 2-7, enhanced by the gene product of sfp,accepted 7-hydroxyheptanoate as substrate as confirmed against the emptyvector control (see FIG. 11), and synthesized 7-hydroxyheptanal.

Example 5 Enzyme Activity of ω-Transaminase for 7-Aminoheptanol, Forming7-Oxoheptanol

A nucleotide sequence encoding an N-terminal His-tag was added to theChromobacterium violaceum, Pseudomonas syringae and Rhodobactersphaeroides genes encoding the ω-transaminases of SEQ ID NOs: 8, 10 and11, respectively (see FIG. 7) such that N-terminal HIS taggedω-transaminases could be produced. The modified genes were cloned into apET21a expression vector under the T7 promoter. Each expression vectorwas transformed into a BL21[DE3] E. coli host. Each resultingrecombinant E. coli strain were cultivated at 37° C. in a 250 mL shakeflask culture containing 50 mL LB media and antibiotic selectionpressure, with shaking at 230 rpm. Each culture was induced overnight at16° C. using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e., 7-aminoheptanolto 7-oxoheptanol) were performed in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 10 mM 7-aminoheptanol, 10mM pyruvate, and 100 μM pyridoxyl 5′ phosphate. Each enzyme activityassay reaction was initiated by adding cell free extract of theω-transaminase gene product or the empty vector control to the assaybuffer containing the 7-aminoheptanol and then incubated at 25° C. for 4h, with shaking at 250 rpm. The formation of L-alanine was quantifiedvia RP-HPLC.

Each enzyme only control without 7-aminoheptanol had low base lineconversion of pyruvate to L-alanine. See FIG. 14.

The gene products of SEQ ID NO 8, 10 & 11 accepted 7-aminoheptanol assubstrate as confirmed against the empty vector control (see FIG. 19)and synthesized 7-oxoheptanol as reaction product. Given thereversibility of the ω-transaminase activity (see Example 2), it can beconcluded that the gene products of SEQ ID 8, 10 & 11 accept7-oxoheptanol as substrate and form 7-aminoheptanol.

Example 6 Enzyme Activity of ω-Transaminase Using Heptamethylenediamineas Substrate and Forming 7-Aminoheptanal

A sequence encoding an N-terminal His-tag was added to theChromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas syringae,Rhodobacter sphaeroides, Escherichia coli, and Vibrio fluvialis genesencoding the ω-transaminases of SEQ ID NOs: 8-13, respectively (see FIG.7) such that N-terminal HIS tagged ω-transaminases could be produced.The modified genes were cloned into a pET21a expression vector under theT7 promoter. Each expression vector was transformed into a BL21[DE3] E.coli host. Each resulting recombinant E. coli strain were cultivated at37° C. in a 250 mL shake flask culture containing 50 mL LB media andantibiotic selection pressure, with shaking at 230 rpm. Each culture wasinduced overnight at 16° C. using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e.,heptamethylenediamine to 7-aminoheptanal) were performed in a buffercomposed of a final concentration of 50 mM HEPES buffer (pH=7.5), 10 mMheptamethylenediamine, 10 mM pyruvate, and 100 μM pyridoxyl 5′phosphate. Each enzyme activity assay reaction was initiated by addingcell free extract of the ω-transaminase gene product or the empty vectorcontrol to the assay buffer containing the heptamethylenediamine andthen incubated at 25° C. for 4 h, with shaking at 250 rpm. The formationof L-alanine was quantified via RP-HPLC.

Each enzyme only control without heptamethylenediamine had low base lineconversion of pyruvate to L-alanine. See FIG. 14.

The gene products of SEQ ID NO 8-13 accepted heptamethylenediamine assubstrate as confirmed against the empty vector control (see FIG. 17)and synthesized 7-aminoheptanal as reaction product. Given thereversibility of the ω-transaminase activity (see Example 2), it can beconcluded that the gene products of SEQ ID 8-13 accept 7-aminoheptanalas substrate and form heptamethylenediamine.

Example 7 Enzyme Activity of Carboxylate Reductase forN7-Acetyl-7-Aminoheptanoate, Forming N7-Acetyl-7-Aminoheptanal

The activity of each of the N-terminal His-tagged carboxylate reductasesof SEQ ID NOs: 3, 6, and 7 (see Examples 4, and FIG. 7) for convertingN7-acetyl-7-aminoheptanoate to N7-acetyl-7-aminoheptanal was assayed intriplicate in a buffer composed of a final concentration of 50 mM HEPESbuffer (pH=7.5), 2 mM N7-acetyl-7-aminoheptanoate, 10 mM MgCl₂, 1 mMATP, and 1 mM NADPH. The assays were initiated by adding purifiedcarboxylate reductase and phosphopantetheine transferase or the emptyvector control to the assay buffer containing theN7-acetyl-7-aminoheptanoate then incubated at room temperature for 20min. The consumption of NADPH was monitored by absorbance at 340 nm.Each enzyme only control without N7-acetyl-7-aminoheptanoatedemonstrated low base line consumption of NADPH. See FIG. 9.

The gene products of SEQ ID NO 3, 6, and 7, enhanced by the gene productof sfp, accepted N7-acetyl-7-aminoheptanoate as substrate as confirmedagainst the empty vector control (see FIG. 12), and synthesizedN7-acetyl-7-aminoheptanal.

Example 8 Enzyme Activity of ω-Transaminase UsingN7-Acetyl-1,7-Diaminoheptane, and Forming N7-Acetyl-7-Aminoheptanal

The activity of the N-terminal His-tagged ω-transaminases of SEQ ID NOs:8-13 (see Example 6, and FIG. 7) for convertingN7-acetyl-1,7-diaminoheptane to N7-acetyl-7-aminoheptanal was assayedusing a buffer composed of a final concentration of 50 mM HEPES buffer(pH=7.5), 10 mM N7-acetyl-1,7-diaminoheptane, 10 mM pyruvate and 100 μMpyridoxyl 5′ phosphate. Each enzyme activity assay reaction wasinitiated by adding a cell free extract of the ω-transaminase or theempty vector control to the assay buffer containing theN7-acetyl-1,7-diaminoheptane then incubated at 25° C. for 4 h, withshaking at 250 rpm. The formation of L-alanine was quantified viaRP-HPLC.

Each enzyme only control without N7-acetyl-1,7-diaminoheptanedemonstrated low base line conversion of pyruvate to L-alanine. See FIG.14.

The gene product of SEQ ID NO 8-13 accepted N7-acetyl-1,7-diaminoheptaneas substrate as confirmed against the empty vector control (see FIG. 18)and synthesized N7-acetyl-7-aminoheptanal as reaction product.

Given the reversibility of the ω-transaminase activity (see example 2),the gene products of SEQ ID 8-13 accept N7-acetyl-7-aminoheptanal assubstrate forming N7-acetyl-1,7-diaminoheptane.

Example 9 Enzyme Activity of Carboxylate Reductase Using PimelateSemialdehyde as Substrate and Forming Heptanedial

The N-terminal His-tagged carboxylate reductase of SEQ ID NO 7 (seeExample 4 and FIG. 7) was assayed using pimelate semialdehyde assubstrate. The enzyme activity assay was performed in triplicate in abuffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5),2 mM pimelate semialdehyde, 10 mM MgCl₂, 1 mM ATP and 1 mM NADPH. Theenzyme activity assay reaction was initiated by adding purifiedcarboxylate reductase and phosphopantetheine transferase or the emptyvector control to the assay buffer containing the pimelate semialdehydeand then incubated at room temperature for 20 min. The consumption ofNADPH was monitored by absorbance at 340 nm. The enzyme only controlwithout pimelate semialdehyde demonstrated low base line consumption ofNADPH. See FIG. 9.

The gene product of SEQ ID NO 7, enhanced by the gene product of sfp,accepted pimelate semialdehyde as substrate as confirmed against theempty vector control (see FIG. 13) and synthesized heptanedial.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1.-32. (canceled)
 33. A recombinant host comprising at least oneexogenous nucleic acid encoding (i) a (homo)_(n)citrate synthaseclassified under EC 2.3.3.14 or EC 2.3.3.13, (ii) a (homo)_(n)citratedehydratase and a (homo)_(n)aconitate hydratase classified under EC4.2.1.114, EC 4.2.1.36, or EC 4.2.1.33, (iii) an iso(homo)_(n)citratedehydrogenase classified under EC 1.1.1.85, EC 1.1.1.87, or EC1.1.1.286, and/or (iv) an indolepyruvate decarboxylase classified underEC 4.1.1.43 or EC 4.1.1.74, or a 2-oxoglutarate dehydrogenase complexcomprising one or more enzymes classified under EC 1.2.4.2, EC 1.8.1.4,and EC 2.3.1.61, said host producing pimeloyl-CoA or pimelatesemialdehyde.
 34. The recombinant host of claim 33, further comprisingat least one exogenous nucleic acid encoding one or more of athioesterase classified under EC 3.1.2.-, an aldehyde dehydrogenaseclassified under EC 1.2.1.3, a 7-oxoheptanoate dehydrogenase classifiedunder EC 1.2.1.-, a 6-oxohexanoate dehydrogenase classified under EC1.2.1.-, a glutaconate CoA-transferase classified under EC 2.8.3.12, areversible succinyl-CoA-ligase classified under EC 6.2.1.5, anacetylating aldehyde dehydrogenase classified under EC 1.2.1.10, or acarboxylate reductase classified under EC 1.2.99.6, said host producingpimelic acid or pimelate semialdehyde.
 35. The recombinant host of claim34, further comprising at least one exogenous nucleic acid encoding aω-transaminase classified under EC 2.6.1.-, said host producing7-aminoheptanoate.
 36. The recombinant host of claim 34, furthercomprising one or more of a 4-hydroxybutyrate dehydrogenase classifiedunder EC 1.1.1.-, a 5-hydroxypentanoate dehydrogenase classified underEC 1.1.1.-, or a 6-hydroxyhexanoate dehydrogenase classified under EC1.1.1.258, said host producing 7-hydroxyheptanoic acid.
 37. Therecombinant host of claim 33, further comprising at least one exogenousnucleic acid encoding a ω-transaminase classified under EC 2.6.1.-, adeacetylase classified under EC 3.5.1.62, a N-acetyl transferaseclassified under EC 2.3.1.32 or an alcohol dehydrogenase classifiedunder EC 1.1.1.-, said host producing heptamethylenediamine.
 38. Therecombinant host of claim 36, further comprising at least one exogenousnucleic acid encoding a (i) carboxylate reductase classified under EC1.2.99.6 enhanced by a phosphopantetheinyl transferase classified underEC 2.7.8.- or (ii) an alcohol dehydrogenase classified under EC 1.1.1.-,said host producing 1,7-heptanediol.